Efficient synthesis of nicotinamide mononucleotide

ABSTRACT

The invention provides a process for the preparation of nicotinamide mononucleotide having formula (I): 
     
       
         
         
             
             
         
       
         
         The method involves the protection of nicotinamide riboside by ketalization, followed by phosphorylation and then deprotection to provide nicotinamide mononucleotide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of U.S. Provisional PatentApplication Nos. 62/139,235, filed Mar. 27, 2015, and 62/115,920, filedMay 1, 2015, which are incorporated by reference.

BACKGROUND OF THE INVENTION

The cellular redox reactions of coenzymes NAD⁺, NADH and NADP⁺, NADPHare well known (Pollak, N. et al, Biochem. J., 402: 205-218 (2007)). Itis known that NAD⁺ plays an important role in apoptosis (Gendron, M. C.et al., Biochem. J., 353: 357 (2001)), calcium mobilization (Guse, A. H.et al., J. Biol. Chem., 280: 15952 (2005)), cell proliferation(Bruzzone, S. et al., Biochem. J., 375: 395 (2003)), aging (Blasco, M.A., Nat. Rev. Genet., 6: 611 (2005)), gene expression (Girolamo, M. D.et al., J. Biol. Chem., 282: 16441 (2007); Sauve, A. A.; Schramm, V. L.,Biochemistry, 42: 9249 (2003); Michan, S. et al., Biochem. J., 404: 1(2007); Nakano, T. et al., Proc. Natl. Acad. Sci. U.S.A., 103: 13652(2006); Culver, G. M. et al., J. Biol. Chem., 272: 13203 (1997); Berger,F. et al., Proc. Natl. Acad. Sci. U.S.A., 104: 3765 (2007)), immunesystem modulation (Song, E. K. et al., Biochem. Biophys. Res. Commun.,367: 156 (2008); Seman, M. et al., Immunity, 19: 571 (2003)), energymetabolism and metabolic regulation. Mono and poly (ADP-ribose)polymerases use NAD⁺as substrate for protein covalent modifications(Ziegler, M., Eur. J. Biochem., 267: 1550 (2000); Guarente, L. et al.,Cell, 120: 473 (2005); Marmorstein, R., Biochem. Soc. Trans., 32: 904(2004); Magni, G. et al., Cell. Mol. Life Sci., 61: 19 (2004); Araki, T.et al., Science, 305: 1010 (2004)). NAD⁺ can be synthesizedenzymatically (Suhadolnik, R. J. et al., Biol. Chem., 252: 4125 (1977))and chemically (Jeck, R. et al., Eds., Academic: New York, 66: 62(1979)) from various precursors of vitamin B₃ (nicotinic acid (NA),nicotinamide (Nam), nicotinamide riboside (NR), nicotinamidemononucleotide (NMN)) and from tryptophan. NAD⁺-tutilizing reactionsliberate nicotinamide, which is recycled to form NMN from nicotinamideand 5-phosphoribosyl pyrophosphate using the enzyme nicotinamidephosphoribosyltransferase. The synthesized NMN reacts with ATP and isconverted to NAD⁺ by nicotinamide mononucleotide adenyltransferase(NMNAT).

Increasing interest in precursors that can be administrated to increasephysiological NAD⁺ provides impetus to develop efficient and practicalsyntheses of precursors such as NR and NMN. A highly efficient chemicalsynthesis of NR has been developed (Yang, T. et al., J. Med. Chem., 50:6458 (2007)). Literature indicates that aside from enzymatic reactionsthere are few different chemical methods for the synthesis ofnicotinamide mononucleotide and derivatives (Burgos, E. S. et al.,Biochemistry, 47: 11086 (2008); Rozenberg, A. et al., J. Org. Chem., 73:9314 (2008)). Existing synthetic strategies involve complicatedintermediates, and isolation of NMN is difficult in good yields.Moreover, enzymatic reactions are typically limited to small scalechemical synthesis and are expensive, and thus are less immediatelyscalable to multigram or kilogram scale.

Thus, there is an unmet need for an improved process for the preparationof nicotinamide mononucleotide.

BRIEF SUMMARY OF THE INVENTION

The invention provides a process for the preparation of nicotinamidemononucleotide having formula (I):

or a salt thereof, wherein the process comprises the steps of:

-   (i) reacting nicotinamide riboside having formula (II):

with a ketalization reagent that is R¹R²C(OR³)(OR⁴) or R¹R²C═O,

-   wherein R¹ and R² are independently C₁-C₆ alkyl or, taken together    along with the carbon atom to which they are attached, form a 5-7    membered carbocyclic or heterocyclic ring, and-   wherein R³ and R⁴ are independently C₁-C₆ alkyl, in a solvent in the    presence of an acid catalyst, to form a compound of formula (III):

-   (ii) isolating the compound of formula (III),-   (iii) reacting the compound of formula (III) with a mixture of POCl₃    and PO(OR⁵)₃, wherein R⁵ is C₁-C₆ alkyl, followed by treatment with    water to form a compound of formula (IV)

-   (iv) isolating the compound of formula (IV),-   (v) reacting the compound of formula (IV) with an acid catalyst in a    solvent or mixture of solvents to provide nicotinamide    mononucleotide, and-   (vi) isolating nicotinamide mononucleotide.

The invention also provides a process for the preparation of a compoundof formula (III):

wherein R¹ and R² are independently C₁-C₆ alkyl or, taken together alongwith the carbon atom to which they are attached, form a 5-7 memberedcarbocyclic or heterocyclic ring, wherein the process comprises the stepof reacting nicotinamide riboside of formula (II):

with a ketalization reagent that is R¹R²C(OR³)(OR⁴) or R¹R²C═O,

-   wherein R¹ and R² are independently C₁-C₆ alkyl or, taken together    along with the carbon atom to which they are attached, form a 5-7    membered carbocyclic or heterocyclic ring, and wherein R³ and R⁴ are    independently C₁-C₆ alkyl, in a solvent in the presence of an acid    catalyst, to form the compound of formula (III).

The invention further provides a process for the preparation of acompound of formula (IV):

or a salt thereof, wherein R¹ and R² are independently C₁-C₆ alkyl or,taken together along with the carbon atom to which they are attached,form a 5-7 membered carbocyclic or heterocyclic ring,

-   wherein the process comprises the step of reacting a compound of    formula (III):

with a mixture of POCl₃ and PO(OR⁵)₃, wherein R⁵ is C₁-C₆ alkyl,followed by treatment with water to form a compound of formula (IV).

The invention additionally provides a process for the preparation ofnicotinamide mononucleotide having formula (I):

or a salt thereof, wherein the process comprises the step of reacting acompound of formula (IV):

wherein R¹ and R² are independently C₁-C₆ alkyl or, taken together alongwith the carbon atom to which they are attached, form a 5-7 memberedcarbocyclic or heterocyclic ring, with an acid catalyst in a solvent ormixture of solvents to provide nicotinamide mononucleotide.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 shows a titration curve for nicotinamide mononucleotide chloride.

FIG. 2 shows the 500 MHz ¹H NMR (500 MHz, D₂O) spectra of zwitterion 4and chloride salt 7 in D₂O superimposed. The peaks labeled “S” for thezwitterions are due to ethanol impurity.

FIG. 3 shows the ³¹P NMR (500 MHz, D₂O) spectrum of zwitterion 4.

FIG. 4 shows the ³¹P NMR (500 MHz, D₂O) spectrum of chloride salt 7.

FIG. 5 shows the overlaid ³¹P NMR spectra of zwitterion 4 and chloridesalt 7.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a process for the preparation of nicotinamidemononucleotide having formula (I):

or a salt thereof, wherein the process comprises the steps of:

-   (i) reacting nicotinamide riboside having formula (II):

with a ketalization reagent that is R¹R²C(OR³)(OR⁴) or R¹R²C═O,

-   wherein R¹ and R² are independently C₁-C₆ alkyl or, taken together    along with the carbon atom to which they are attached, form a 5-7    membered carbocyclic or heterocyclic ring, and wherein R³ and R⁴ are    independently C₁-C₆ alkyl, in a solvent in the presence of an acid    catalyst, to form a compound of formula (III):

-   (ii) isolating the compound of formula (III),-   (iii) reacting the compound of formula (III) with a mixture of POCl₃    and PO(OR⁵)₃, wherein R⁵ is C₁-C₆ alkyl, followed by treatment with    water to form a compound of formula (IV):

-   (iv) isolating the compound of formula (IV),-   (v) reacting the compound of formula (IV) with an acid catalyst in a    solvent or mixture of solvents to provide nicotinamide    mononucleotide, and-   (vi) isolating nicotinamide mononucleotide.

In an embodiment, nicotinamide mononucleotide is synthesized as shown inScheme 1.

Nicotinamide riboside 1, wherein X⁻ is an anion, is reacted with aketalization reagent that is R¹R²C(OR³)(OR⁴) or R¹R²C═O, wherein R¹ andR² are independently C₁-C₆ alkyl or, taken together along with thecarbon atom to which they are attached, form a 5-7 membered carbocyclicor heterocyclic ring, and wherein R³ and R⁴ are independently C₁-C₆alkyl, in a solvent in the presence of an acid catalyst, to formcompound 5. In a preferred embodiment, the ketalization reagent isR¹R²C(OR³)(OR⁴) and more preferably is 2,2-dimethoxypropane.

It will be understood that, when a compound is shown as a cation withouthaving an anion, the positive charge on the cation can be countered byany suitable anion or anionic component having a negative charge. Theanion can be any suitable organic, inorganic, or polymeric anion withoutlimitation. In an embodiment, the anion is trifluoromethanesulfonate.

The acid catalyst can be any suitable acid catalyst, for example, theacid catalyst can be an inorganic acid catalyst such as sulfuric acid,hydrochloric acid, phosphoric acid, and the like. The acid catalyst canbe an organic acid catalyst, for example, p-toluenesulfonic acid,methylsulfonic acid, trifluoromethylsulfonic acid, and the like. In apreferred embodiment, the acid catalyst is sulfuric acid, morepreferably concentrated sulfuric acid.

The solvent can be any suitable solvent and can be, for example,acetonitrile, dichloromethane, acetone, dimethylformamide,dimethylsulfoxide, and the like. Preferably, the solvent isacetonitrile.

The ketalization can be conducted at any temperature. For example, theketalization can be conducted at about 0° C. to about 50° C. Preferably,the ketalization is conducted starting at about 0° C. followed bywarming to room temperature.

Compound 5 can be optionally isolated by quenching the reaction mixturewith a base such as sodium carbonate, followed by filtration and thenevaporation of solvents. In other embodiments, the reaction mixture canbe partitioned between water and an organic solvent such asdichloromethane, ethyl acetate, and the like. The acid catalyst can beneutralized before partitioning or can be quenched in an aqueoussolution of a base followed by extraction with a solvent. Compound 5 canbe isolated by silica gel chromatography or by crystallization.

Compound 5 can be phosphorylated using any suitable conditions, forexample, compound 5 can be phosphorylated in a mixture of phosphorusoxychloride and PO(OR⁵)₃, wherein R⁵ is C₁-C₆ alkyl. Preferably,compound 5 is phosphorylated in a mixture of phosphorus oxychloride andtriethylphosphate to provide compound 6. The phosphorylation can beconducted at any suitable temperature. For example, the phosphorylationcan be conducted at about 0° C. to about 50° C. and is preferablyconducted at 0° C.

Compound 6 can be optionally isolated by quenching the reaction mixturewith a base such as sodium carbonate, followed by extraction of excessunreacted triethylphosphate with a solvent such as ethyl acetate andthen recovery of compound 6 from the aqueous layer by evaporation.Compound 6 can be isolated by silica gel chromatography or bycrystallization.

Nicotinamide mononucleotide 4 is obtained by deprotection of compound 6via acid catalyzed deketalization. The deprotection can be conducted inan aqueous solvent mixture, for example, in a mixture of dichloromethaneand water. The deprotection can be conducted in a nonaqueous solvent.For example, the deprotection can be conducted in a hydroxylic solventsuch as methanol or ethanol, preferably in methanol. The acid catalystcan be any suitable acid catalyst as described herein in connection withthe preparation of compound 5, and is preferablytrifluoromethanesulfonic acid or concentrated hydrochloric acid.

Nicotinamide mononucleotide can be isolated using any suitable isolationprocedure. For example, the reaction mixture can be at least partiallyevaporated to remove volatile organic solvent, and the residue can betreated with water and then neutralized to pH 5-6 with a base such assodium carbonate. The crude product can be purified in any suitablemanner to provide purified nicotinamide mononucleotide. For example, thecrude product can be purified using reverse phase chromatography on aC18 column with water as eluent to provide purified nicotinamidemononucleotide.

Nicotinamide mononucleotide of formula (I) and the compound of formula(IV) can be in the form of a zwitterion or any suitable salt thereof.For example, nicotinamide mononucleotide of formula (I) and the compoundof formula (IV) can be in the form of a protonated salt or a monobasicsalt thereof. As used herein, the term protonated salt refers to thecompounds of formulas (I) and (IV) wherein the phosphate (—O—P(═O)(OH)₂group is not ionized. As used herein, the term monobasic salt refers tothe compounds of formulas (I) and (IV) wherein the phosphate(—O—P(═O)(O⁻)₂ group is fully ionized. Illustrative embodiments ofzwitterions of nicotinamide mononucleotide and the compound of formula(IV) are:

Examples of protonated salts of nicotinamide mononucleotide of formula(I) and the compound of formula (IV) include:

wherein X⁻ can be any suitable monovalent anion.

Examples of a monobasic salt of nicotinamide mononucleotide of formula(I) and the compound of formula (IV) include:

wherein M⁺ can be any suitable monovalent cation. In other embodiments,the monobasic salt can be associated with any suitable divalent cationM²⁺, as illustrated for nicotinamide mononucleotide:

The salts can be prepared by reacting the zwitterionic forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two.Generally, nonaqueous media such as ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred. Lists of suitable salts arefound in Remington's Pharmaceutical Sciences, 18th ed., Mack PublishingCompany, Easton, Pa., 1990, p. 1445, and Journal of PharmaceuticalScience, 66, 2-19 (1977).

Suitable bases include inorganic bases such as alkali and alkaline earthmetal bases, e.g., those containing metallic cations such as sodium,potassium, magnesium, calcium and the like. Non-limiting examples ofsuitable bases include sodium hydroxide, potassium hydroxide, sodiumcarbonate, and potassium carbonate. Suitable acids include inorganicacids such as hydrochloric acid, hydrobromic acid, hydroiodic acid,sulfuric acid, phosphoric acid, and the like, and organic acids such asp-toluenesulfonic, methanesulfonic acid, benzenesulfonic acid, oxalicacid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citricacid, benzoic acid, acetic acid, maleic acid, tartaric acid, fattyacids, long chain fatty acids, and the like. In embodiments, theprotonated and monobasic salts comprise pharmaceutically acceptablesalts. Preferred monobasic salts include sodium and potassium salts.Preferred protonated salts include hydrochloride and hydrobromide salts.

In embodiments, the protonated salt is produced during the conversion ofcompound 6 to compound 4. The protonated salt 7 and zwitterion 4 can beproduced as shown in Scheme 2:

The following example further illustrates the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE 1

This example demonstrates a process for the synthesis of nicotinamidemononucleotide riboside, in accordance with an embodiment of theinvention.

Step 1: Preparation of3-Carbamoyl-1-((3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyridin-1-ium)(2)

In a flame dried flask under an argon atmosphere, concentrated sulfuricacid (22 μL, 0.40 mmol) was slowly added to dry acetonitrile (2.0 mL) at0° C. After 5 minutes, 2,2-dimethoxypropane (0.6 mL, excess) was addedto the stirred acetonitrile solution at the same temperature.Nicotinamide riboside solid (1) (250 mg, 0.61 mmol) was added to thereaction mixture at 0° C., and the reaction was immediately warmed to25° C. over 10 min. The progress of the reaction was monitored by thinlayer chromatography (TLC) and high performance liquid chromatography(HPLC). HPLC showed 95% of starting material was consumed after 10 min.The reaction mixture was cooled again to 0° C. in an ice bath, and wasquenched by addition of powdered solid Na₂CO₃ (50 mg 0.47 mmol) andstirred for 5 min. 0.1 mL water was added slowly to improveneutralization of the acid. Residual solids were filtered, and thefiltrate (acetonitrile) was evaporated under high vacuum to obtain thecrude product. The crude product was dissolved in a minimum volume ofDCM and was purified by silica gel column (60 A^(o) using DCM/MeOH (9:1)to obtain 2 as a white solid. Yield 96%.

¹H NMR (CD₃OD, 500 MHz): δ 9.56 (s, 1H, H-2), 9.31 (d, 1H, J=6.3 Hz,H-6), 9.02 (d, 1H, J=7.8 Hz, H-4), 8.25 (t, 1H, J=7.6 Hz, H-5), 6.41 (s,1H, H-4′), 5.22-5.19 (m, 1H, H-1′), 4.99 (d, 1H, J=5.7 Hz, H-2′),3.99-3.94 (dd, 1H, J=1.8 and 12.3, H-3′), 3.82-3.77 (dd, 1H, J=2.3 and11.6 Hz, H-5′ a), 3.70-3.68 (m, 1H, H-5′b), 1.66 (s, 3H, —CH₃), 1.45 (s,3 H, —CH₃). ¹³C NMR (CD₃OD, 125 MHz): 142.7, 142.4, 140.5, 133.8, 127.4,114.0, 103.9, 90.4, 87.7, 82.3, 61.3, 34.0, 25.9, 23.9.

Step 2: Preparation of3-Carbamoyl-1-((3aR,4R,6R,6aR)-2,2-dimethyl-6-((phosphonooxy)methyl)tetrahydrofuro[3,4-d][1,3] dioxol-4-yl)pyridin-11-ium) (3)

Compound 2 (880 mg, 2 mmol) was added to 8 mL of dry triethyl phosphateat 0° C. under an argon atmosphere in a flame-dried flask. After 10minutes, 470 μL phosphorous oxychloride (2.5 eq, 5 mmol) was slowlyadded portion-wise (176 μL+176μL+118 μL) to the stirred and chilledtriethyl phosphate solution. The mixture was stirred at 0 ° C. for 48hours. Progress of the reaction was monitored by HPLC, which showed that75% of starting material was consumed after 48 h, with concomitantincrease in product 3 which eluted at a shorter retention time. Thereaction mixture was cooled to 0° C. and was quenched with 3 consecutive1 mL portions of cold saturated Na₂CO₃ solution until the acid wasneutralized and bubbling ceased. Triethyl phosphate was removed byextraction with ethyl acetate (3×10 mL). The combined ethyl acetatelayers were then extracted with water (3×5 mL) to remove the crudeproduct. The combined water layers and initial water layer containingproduct 3 were dried under high vacuum to obtain the crude product. Thecrude product was dissolved in a minimum volume of 9:1 DCM:methanol asneeded to solubilize, and the crude product was purified by columnchromatography on silica gel using DCM/MeOH (6:4) to provide (3) as awhite solid. Yield 80%.

¹H NMR (D₂O, 500 MHz): δ 9.31 (s, 1H, H-2), 9.11 (d, 1H, J=6.4 Hz, H-6),8.86 (d, 1H, J=8.2 Hz, H-4), 8.22-8.18 (m, 1H, H-5), 6.37 (d, 1H, J=2.5Hz, H-4′), 5.30 (dd, 1H, J=2.6 and 5.8 Hz, H-1′), 5.07 (d, 1H, J=5.7 Hz,H-2′), 4.99-4.97 (m, 1H, H-3′), 4.18 (t, 1H, J=2.1 Hz, H-5′a), 4.16 (t,1H, J=2.1 Hz, H-5′b), 1.60 (s, 3H, -CH₃), 1.41 (s, 3 H, —CH₃). ¹³C NMR(D₂O, 125 MHz): 150.8, 145.7, 139.5, 128.2, 103.2, 88.2, 86.8, 82.4,65.2, 30.0, 25.9, 24.2.

Step 3: Preparation of3-Carbamoyl-1-((2R,5R)-3,4-dihydroxy-5-((phosphonooxy)methyl)tetrahydrofuran-2-yl)pyridin-1-ium)(4)

(A) Deprotection in dichloromethane/water:

TFA (0.6 mL, 7.8. mmol) was slowly added to a solution of compound 3(1000 mg, 2.67 mmol) in 30 mL dichloromethane and water (1:1) at 0° C.The reaction mixture was stirred vigorously and allowed to warm to roomtemperature. After 16 hours (reaction progress was monitored by HPLC,which showed that 95% of starting material was consumed after 16 h), thereaction mixture was evaporated. Residual water was neutralized withminimal Na₂CO₃ to pH 6, and the water layer was evaporated to obtain thecrude product. The crude product was dissolved in minimum water and waspurified by a C18 column using water to provide NMN (4) as a whitesolid. Yield 90%.

¹H NMR (D₂O, 500 MHz): δ 9.39 (s, 1H, H-2), 9.21 (d, 1H, J=6.2 Hz, H-6),8.90 (d, 1H, J=8.1 Hz, H-4), 8.22 (t, 1H, J=6.9 Hz, H-5), 6.14 (d, 1H, J=5.6 Hz, H-4′), 4.56 (t, 1H, J=2.4 Hz, H-1′), 4.48 (t, 1H, J=5.3 Hz,H-2′), 4.38-4.35 (m, 1H, H-3′), 4.25-4.20 (m, 1H, H-5′a); 4.01-4.05 (m,1 H, H-5′b). ¹³C NMR (D₂O,125 MHz): 165.9, 146.0, 142.5, 139.9, 134.0,128.5, 100.0, 87.5, 77.8, 75.1, 64.2.

(B) Deprotection in methanolic HCl:

Concentrated hydrochloric acid (672 μL, 8.06 mmol) was slowly added to asolution of compound 3 (1000 mg, 2.67 mmol) in 30 mL of methanol underan argon atmosphere in a flame dried flask. The reaction mixture wasallowed to warm to room temperature. After 40 hours HPLC showed nostarting material remained. The reaction mixture was then evaporated todryness, and 5 mL water was added to dissolve solids. Solid Na₂CO₃ wasadded to adjust pH to 5. NMN was purified on a C18 column using water aseluent to provide NMN (4) as a white solid. Yield 67%.

¹H NMR (D₂O, 500 MHz): δ 9.39 (s, 1H, H-2), 9.21 (d, 1H, J=6.2 Hz, H-6),8.90 (d, 1H, J=8.1 Hz, H-4), 8.22 (t, 1H, J=6.9 Hz, H-5), 6.14 (d, 1H,J=5.6 Hz, H-4′), 4.56 (t, 1H, J=2.4 Hz, H-1′), 4.48 (t, 1H, J=5.3 Hz,H-2′), 4.38-4.35 (m, 1H, H-3′), 4.25-4.20 (m, 1H, H-5′a); 4.01-4.05 (m,1 H, H-5′b). ¹³C NMR (D₂O, 125 MHz): 165.9, 146.0, 142.5, 139.9, 134.0,128.5, 100.0, 87.5, 77.8, 75.1, 64.2.

EXAMPLE 2

This example demonstrates the titration of nicotinamide mononucleotidechloride salt with sodium hydroxide.

Nicotinamide mononucleotide-chloride salt (110 mg, 0.29 mmol) wasdissolved in 2 ml water (approximate concentration 150 mM). To theresulting solution 200 μL of 150 mM NaOH solution (0.1 eq) was added andpH was measured by pH meter. Addition of 200 μL of 150 mM NaOH solutionwas continued and the pH measured and plotted. 2 pKa values weredetermined by this titration, namely the first pK_(a1)=2.1 for theconversion of [NMNH]Cl to the neutral zwitterion form and thenpK_(a2)=6.7 to form the mono anion [NMN]⁻ (Scheme 3). The titration ofboth protons indicates the successful stabilization and characterizationof the cation [NMNH]Cl form of NMN.

The titration curve is shown in FIG. 1.

EXAMPLE 3

This example shows the ¹H NMR and ³¹P NMR spectra of zwitterion 4 andchloride salt 7.

FIG. 2 shows the 500 MHz ¹H NMR (500 MHz, D₂O) spectra of zwitterion 4and chloride salt 7 in D₂O superimposed.

FIG. 3 shows the ³¹P NMR 500 MHz, D₂O) spectrum of zwitterion 4.

FIG. 4 shows shows the ³¹P NMR 500 MHz, D₂O) spectrum of chloride salt7.

FIG. 5 shows the overlaid ³¹P NMR spectra of zwitterion 4 and chloridesalt 7.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A process for the preparation of nicotinamide mononucleotide havingformula (I):

or a salt thereof, wherein the process comprises the steps of: reactingnicotinamide riboside having formula (II):

with a ketalization reagent that is R¹R²C(OR³)(_(OR) ⁴) or R¹ R²C═O,wherein R¹ and R² are independently C1-C6 alkyl or, taken together alongwith the carbon atom to which they are attached, form a 5-7 memberedcarbocyclic or heterocyclic ring, and wherein R³ and R⁴ areindependently C₁-C₆ alkyl, in a solvent in the presence of an acidcatalyst, to form a compound of formula (III):

(ii) isolating the compound of formula (III), (iii) reacting thecompound of formula (III) with a mixture of POCl₃ and PO(OR⁵)₃, whereinR⁵ is C₁-C₆ alkyl, followed by treatment with water to form a compoundof formula (IV):

or a salt thereof, (iv) isolating the compound of formula (IV), (v)reacting the compound of formula (IV) with an acid catalyst in a solventor mixture of solvents to provide nicotinamide mononucleotide, and (vi)isolating nicotinamide mononucleotide.
 2. (canceled)
 3. The process ofclaim 1, wherein the ketalization reagent is (CH₃)₂C(OCH₃)₂.
 4. Theprocess of claim 1, wherein the solvent in step (i) is CH₃CN.
 5. Theprocess of claim 4, wherein the acid catalyst in step (i) is H₂SO₄.Attorney Docket 730844 Preliminary Amendment
 6. The process of any oncof claim 1, wherein the solvent in step (v) is a mixture ofdichloromethane and water.
 7. The process of claim 1, wherein thesolvent in step (v) is methanol.
 8. The process of claim 1, wherein theacid catalyst in step (v) is trifluoroacetic acid.
 9. The process ofclaim 1, wherein the acid catalyst in step (v) is hydrochloric acid. 10.A process for the preparation of a compound of formula (III):

wherein R¹ and R² are independently C₁-C₆ alkyl or, taken together alongwith the carbon atom to which they are attached, form a 5-7 memberedcarbocyclic or heterocyclic ring, wherein the process comprises the stepof reacting nicotinamide riboside of formula (II):

with a ketalization reagent that is R¹R²C(OR³)(OR⁴) or R¹R²C═O, whereinR¹ and R² are independently C¹-C⁶ alkyl or, taken together along withthe carbon atom to which they are attached, form a 5-7 memberedcarbocyclic or heterocyclic ring, and wherein R³ and R⁴ areindependently C¹-C⁶ alkyl, in a solvent in the presence of an acidcatalyst, to form the compound of formula (III). wherein the acidcatalyst is selected from the group consisting of sulfuric acid,hydrochloric acid, phosphoric acid, p-toluenesulfonic acid, andtrifluoroacetic acid.
 11. (canceled)
 12. The process of claim 10,wherein the ketalization reagent is (CH₃)₂C(OCH₃)₂.
 13. The process ofclaim 10, wherein the solvent is CH₃CN.
 14. The process of any one ofclaim 10, wherein the acid catalyst is H₂SO₄.
 15. A process for thepreparation of a compound of formula (IV):

or a salt thereof, wherein R¹ and R² are independently C1-C6 alkyl or,taken together along with the carbon atom to which they are attached,form a 5-7 membered carbocyclic or heterocyclic ring, wherein theprocess comprises the step of reacting a compound of formula (III):

with a mixture of POCl₃ and PO(OR⁵)_(3,) wherein R⁵ is C₁-C₆ alkyl,followed by treatment with water to form a compound of formula (IV). 16.A process for the preparation of nicotinamide mononucleotide havingformula (I):

or a salt thereof, wherein the process comprises the step of reacting acompound of formula (IV):

wherein R¹ and R² are independently C₁-C₆ alkyl or, taken together alongwith the carbon atom to which they are attached, form a 5-7 memberedcarbocyclic or heterocyclic ring, with an acid catalyst in a solvent ormixture of solvents to provide nicotinamide mononucleotide, wherein theacid catalyst is selected from the group consisting of sulfuric acid,hsydrochloric acid, phosphoric acid, p-toluenesulfonic acid, andtrifluoroacetic acid.
 17. The process of claim 16, wherein the solventin step (v) is a mixture of dichloromethane and water.
 18. The processof claim 16, wherein the solvent in step (v) is methanol.
 19. Theprocess of claim 16, wherein the acid catalyst is trifluoroacetic acid.20. The process of claim 16, wherein the acid catalyst is hydrochloricacid.
 21. A process for the preparation of nicotinamide mononucleotidehaving formula (I):

or a salt thereof, wherein the process comprises the step of reacting acompound of formula (IV):

wherein R¹ and R² are independently C₁-C₆ alkyl or, taken together alongwith the carbon atom to which they are attached, form a 5-7 memberedcarbocyclic or heterocyclic ring, with an acid catalyst in a solvent ormixture of solvents to provide nicotinamide mononucleotide, wherein thesolvent or mixture of solvents is methanol, ethanol, or a mixture ofdichloromethane and water.
 22. The process of claim 21, wherein the acidcatalyst is trifluoroacetic acid or hydrochloric acid.